1. Field of the Invention
This invention relates to antibody purification techniques. In particular, the invention relates to synthetic human chorionic gonadotropin (hCG) peptides for the purification of polyclonal anti-hCG antibodies.
2. Description of Related Art
Various analytical procedures and devices are commonly employed in assays to determine the presence and/or concentration of substances of interest or clinical significance which may be present in biological fluids or other materials. Such substances are commonly termed "analytes" and can include antibodies, antigens, drugs, hormones, etc. The detection of particular analytes in biological fluids such as serum, plasma, urine, spinal fluid and the like has in recent years become of critical importance in both research and clinical settings. The detection of analytes of interest can often be related to various disease states and consequently is extremely useful in the diagnosis of disease and in monitoring the effectiveness of therapy. When the analytes are antigens or antibodies, assays typically depend upon the immunological reactivity which characterizes these substances. Generally, such assays are collectively termed immunoassays.
Immunoassay techniques take advantage of the mechanisms of the immune systems of higher organisms, wherein antibodies are produced in response to the presence of antigens which are pathogenic or foreign to the organisms. One or more antibodies are produced in response to and are capable of reacting with a particular antigen, thereby creating a highly specific reaction mechanism which can be used in vitro to determine the presence or concentration of that antigen in a biological sample.
The results of an immunoassay, however, can vary according to the type of antibody used and the antibody's specificity for the particular antigen. In a quantitative assay for hCG, the particular anti-hCG antibody used may cross-react with a second glycoprotein antigen, such as human leutenizing hormone (hLH), follicle stimulating hormone (FSH) or thyroid stimulating hormone (TSH), thereby decreasing the accuracy of the assay. For example, an antibody that recognizes and binds with hCG generally will also bind to LH, FSH and TSH rather than specifically binding with hCG. Antibody purification methods have been devised to minimize cross-reactivity. Typical of such conventional methodologies is one which uses a two column technique involving a first affinity column containing a whole molecule hCG to concentrate a specific group of IgG molecules, and a second affinity column containing LH or FSH in an attempt to remove antibodies that cross-react with hCG. The hCG polyclonal antibodies obtained from this conventional procedure still have significant cross-reactivity. In addition, the two column technique is a time consuming and expensive procedure.
Chemical analysis of analytes, such as hCG and hLH, has shown that the cross-reactivity of these hormones is due to their .alpha.-subunits which are structurally similar. Therefore, attempts have been made to separate and purify the .beta.-subunit of hCG (.beta.-hCG) which is comparatively distinct in structure from hLH. The separation procedures, however, can still result in the occurrence of impurities and the inclusion of amino acid sequences common to both hCG and hLH.
The peptide moiety situated at the COOH terminus of .beta.-hCG consists of about 45 amino acids. It was found that this moiety permits a distinct identification of hCG versus hLH (Matsuura et al, Endocrinology 104: 396, 1979). Iwasa et al, describe the production of a synthetic peptide sequence which reproduces a specific portion of the COOH terminal peptide (CTP) of .beta.-hCG for use in the preparation of an anti-hCG antibody (U.S. Pat. No. 4,517,290). The amino acid sequence of the CTP of .beta.-hCG consists of 45 amino acids, as follows: EQU Gly-Gly-Pro-Lys-Asp.sup.105 -His-Pro-Leu-Thr-Cys.sup.110 -Asp-Asp-Pro-Arg-Phe.sup.15 -Gln-Asp-Ser-Ser-Ser.sup.120 -Ser-Lys-Ala-Pro-Pro.sup.125 -Pro-Ser-Leu-Pro-Ser.sup.130 -Pro-Ser-Arg-Leu-Pro.sup.135 -Gly-Pro-Ser-Asp-Thr.sup.140 -Pro-Ile-Leu-Pro-Gin.sup.145
as described by Morgan et al, Mol. Cell. Biochem. 2(1), 97-99 (1973). The synthetic peptide sequence described by iwasa for polyclonal antibody purification comprises CTP fragments or subsequences of from 10 to 23 amino acid residues, such as: EQU Gly-Pro-Ser-Asp-Thr.sup.140 -Pro-Ile-Leu-Pro-Gln.sup.145
with increased sequence lengths up to
Ala-Pro-Pro.sup.125 -Pro-Ser-Leu-Pro-Ser.sup.130 -Pro-Ser-Arg-Leu-Pro.sup.135 -Gly-Pro-Ser-Asp-Thr.sup.140 -Pro-Ile-Leu-Pro-Gln.sup.145.
None of the art described above discloses or suggests the use of CTP sequences greater than 23 amino acid residues in size to alleviate the cross-reactivity characteristics of antibodies.